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Kolthoff Lectureship #3: Professor Carlos J. Bustamante

Izaak M. Kolthoff Lectureship in Chemistry

"Division of Labor and Coordination Among the Subunits of a Viral Ring ATPase"

As part of their infection cycle, many viruses must package their newly replicated genomes inside a protein capsid. Bacteriophage phi29 packages its 6.6 mm long double-stranded DNA using a pentameric ring nano motor that belongs to the ASCE (Additional Strand, Conserved E) superfamily of ATPases. A number of fundamental questions remain as to the coordination of the various subunits in these multimeric rings. The portal motor in bacteriophage phi29 is ideal to investigate these questions and is a remarkable machine that must overcome entropic, electrostatic, and DNA bending energies to package its genome to near-crystalline density inside the capsid. Using optical tweezers, we find that this motor can work against loads of up to ~55 picoNewtons on average, making it one of the strongest molecular motors ever reported. We establish the force-velocity relationship of the motor. Interestingly, the packaging rate decreases as the prohead fills, indicating that an internal pressure builds up due to DNA compression attaining the value of ~6 MegaPascals at the end of the packaging. This pressure, we show, is used as part of the mechanism of DNA injection in the next infection cycle. We have used high-resolution optical tweezers to characterize the steps and intersubunit coordination of the pentameric ring ATPase responsible for DNA packaging in bacteriophage Phi29. By using non-hydrolyzable ATP analogs and stabilizers of the ADP bound to the motor, we establish where DNA binding, hydrolysis, and phosphate and ADP release occur relative to translocation. We show that while only 4 of the subunits translocate DNA, all 5 bind and hydrolyze ATP, suggesting that the fifth subunit fulfills a regulatory function. Finally, we show that the motor not only can generate force but also torque. We characterize the role played by the special subunit in this process and identify this the symmetry-breaking mechanism. These results represent the most complete studies done to date on these ubiquitous class of ring nano motors.

Professor Bustamante

Professor Bustamante is a professor of chemistry, physics, and molecular and cell biology, and the Raymond and Beverly Sackler Professor in Biophysics at the University of California, where he has been a faculty member and researcher since 1998. He received his bachelor’s degree from the Universidad Peruana Cayetano Heredia in Lima, Peru, his master's degree in biochemistry from the Universidad Nacional Mayor de San Marcos in Lima, and his doctorate in biophysics from University of California, Berkeley, where he studied with Ignacio Tinoco Jr. As a post-doctoral fellow at the Lawrence Berkeley National Laboratory, Bustamante studied with Marcos Maestre. Before moving to Berkeley, he was a professor at the University of Mexico, and professor of chemistry and member of the Insitute of Molecular Biology at the University of Oregon. He also was a Howard Hughes Medical Institute investigator. He is a Fellow of the American Physical Society, Fellow of the National Academy of Sciences, and Howard Hughes Medical Institute Investigator.

Research

Professor Bustamante’s laboratory specializes in single molecule biophysics, where his researchers can track and measure the activity of individual enzymes. By looking at each enzyme, they can parse out effects which are not resolvable in bulk experiments. The researchers use optical tweezers, magnetic tweezers, atomic force microscopy, single molecule fluorescence, fluorescence correlation spectroscopy, and super-resolution photo-activatable light microscopy (PALM). They are interested in studying how the cell converts chemical energy into mechanical work through highly specialized molecular machines. The generation, transduction, and regulation of force are key to many central processes in the cell. Many enzymes, such as polymerases, are motors which move along a cellular track, using chemical energy to take regulated steps and control synthesis. These techniques are used to understand:

DNA packaging into bacteriophage Φ29 capsids;

transcription;

translation and protein folding;

protein degradation by ClpXP;

PALM-mitochondrial fission, and

catalysis-enhanced enzyme diffusion

His group is affiliated with the departments of Molecular and Cell Biology, Physics, and Chemistry as well as the Physical Biosciences Division at the Lawrence Berkeley National Laboratory.

About Professor Kolthoff

Izaak Maurits Kolthoff was born on February 11, 1894, in Almelo, Holland. He died on March 4, 1993, in St. Paul, Minnesota. In 1911, he entered the University of Utrecht, Holland. He published his first paper on acid titrations in 1915. On the basis of his world-renowned reputation, he was invited to join the faculty of the University of Minnesota’s Department of Chemistry in 1927. By the time of his retirement from the University in 1962, he had published approximately 800 papers. He continued to publish approximately 150 more papers until his health failed. His research, covering approximately a dozen areas of chemistry, was recognized by many medals and memberships in learned societies throughout the world, including the National Academy of Sciences and the Nichols Medal of the American Chemical Society. Best known to the general public is his work on synthetic rubber. During World War II, the government established a comprehensive research program at major industrial companies and several universities, including Minnesota. Kolthoff quickly assembled a large research group and made major contributions to the program. Many of Kolthoff’s graduate students went on to successful careers in industry and academic life and, in turn, trained many more. In 1982, it was estimated that approximately 1,100 Ph.D. holders could trace their scientific roots to Kolthoff. When the American Chemical Society inaugurated an award for excellence in 1983, he was the first recipient.